Characterization of the molecular basis for virus attachment to cells has importance both for understanding virus tropism and for developing agents that inhibit virus binding or alter the specificity of binding. Recently, a 10 cellular receptor for adenovirus type 2 and other closely related serotypes was identified. This receptor, encoded by a single gene on human chromosome 21 (Mayr et al., J. Virol. 71: 412-8 (1997)), is a 46 kD glycoprotein which also serves as a receptor for group B coxsackieviruses (CBV) and thus was termed CAR. CAR mRNA is present in varying abundance in many human tissues. A broad tissue distribution of CAR protein expression correlates with the broad tropism of CBV, but subgroup C adenoviruses that are known to bind CAR have a much more restricted tropism limited primarily to the upper respiratory tract. Thus, other factors in addition to receptor availability clearly have important roles in determining adenovirus tropism. Although adenovirus binds to CAR with high affinity (Mayr et al., J. Virol. 71: 412-8 (1997); Wickham et al., Cell. 73: 309-19 (1993)), virus titers are significantly reduced on cells with down-regulated CAR expression (Freimuth, P., J. Virol. 70: 4081-5 (1996)). These results suggest that adenovirus infection in vivo may be restricted to cells which express CAR at levels above a minimum threshold concentration. CAR protein levels are relatively low on the apical surface of differentiated (ciliated) respiratory epithelial cell cultures, which may account for the poor efficiency of Gadenoviral gene transfer to human lung tissue in vivo.
Adenovirus binding to CAR results from an interaction between rod-shaped proteins located at the capsid vertices, called viral fibers, and the extracellular region of CAR. The distal, carboxy-terminal end of fiber consists of a globular domain, termed the knob, which has receptor-binding activity. The knob domain of adenovirus type 5 (Ad5) was expressed in E. coli as a soluble, trimeric, biologically active protein, and its 3-dimensional structure was determined by x-ray crystallography. The predicted amino acid sequence of CAR suggests a structure consisting of two extracellular domains related to the immunoglobulin IgV and IgC2 domain folds (Bork et al., J. Mol Biol. 242: 309-20 (1994); Bergelson et al., Science 275: 1320-3 (1997); Tomko et al., Proc. Natl. Acad. Sci. USA 94: 3352-6 (1997)), a single membrane-spanning region, and one carboxy-terminal cytoplasmic domain. Regions of CAR necessary for binding the fiber knob domain have not yet been determined.
In one aspect, the present invention relates to an isolated polypeptide comprising an amino acid sequence which corresponds to adenovirus binding domain D1 of human CAR (coxsackievirus and adenovirus receptor) protein. The present invention also relates to an isolated polypeptide comprising an amino acid sequence which corresponds to extracellular domains D1 and D2 of human CAR protein, which also demonstrates adenovirus binding activity. In other aspects, the invention relates to nucleic acid sequences encoding the D1 and combined D1 and D2 domains. The invention also relates to expression vectors which encode the domains as well as bacterial cells containing such vectors. In a preferred embodiment the D1 polypeptide sequence is fused to a polypeptide sequence which facilitates folding of D1 into a functional, soluble domain when expressed in bacteria.
In another embodiment, the invention relates to a therapeutic method for treating a patient infected with a virus which binds to D1, such as adenoviruses from subgroups A and C, or coxsackievirus subgroup B. The method involves providing a therapeutic composition comprising. D1 and administering it to a patient. Administration is generally topical and to a localized region. Areas of localized infection suitable for treatment include the ocular region, the upper respiratory tract and the gastrointestinal region.
Also encompassed within the scope of the invention are methods based on the experiments described in the Exemplification section set forth below. These include, for example, methods for identifying a protein, and its binding domain, which binds D1, and also a method for identifying an antiviral compound which interferes with viral attachment. Also included is a method for specifically targeting a cell for infection by a virus which binds to D1. This method has practical use in gene therapy, which often utilizes adenovirus expression vectors.